The equilibrium binding and kinetics of assembly of the DNA-dependent RNA polymerase (RNAP) σN-holoenzyme has been investigated using biosynthetically labelled 7-azatryptophyl- (7AW)σN. The spectroscopic properties of such 7AW proteins allows their absorbance and fluorescence to be monitored selectively, even in the presence of high concentrations of other tryptophan-containing proteins. The 7AWσN retained its biological activity in stimulating transcription from σN-specific promoters, and in in vitro gel electrophoresis assays of binding to core RNAP from Escherichia coli. Furthermore, five Trp → Ala single mutants of σN were shown to support growth under conditions of nitrogen limitation, and showed comparable efficiency in activating the σN-dependent nifH promoter in vivo, indicating that none of the tryptophan residues were essential for activity. The equilibrium binding of 7AWσN to core RNAP was examined by analytical ultracentrifugation. In sedimentation equilibrium experiments, absorbance data at 315nm (which reports selectively on the distribution of free and bound 7AWσN) established that a 1:1 complex was formed, with a dissociation constant lower than 2µM. The kinetics of the interaction between 7AWσN and core RNAP was investigated using stopped-flow spectrofluorimetry. A biphasic decrease in fluorescence intensity was observed when samples were excited at 280nm, whereas only the slower of the two phases was observed at 315nm. The kinetic data were analysed in terms of a mechanism in which a fast bimolecular association of σN with core RNAP is followed by a relatively slow isomerization step. The consequences of these findings on the competition between σN and the major sigma factor, σ70, in Escherichia coli are discussed.

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